Biofuels including alcohol or lipid and oil based products that are derived from biological sources are gaining wide consumer and regulatory acceptance as renewable fuels. Widespread commercialization of oils extracted from plant and animal materials (primarily, but not limited to seed oils) as well as alcohols (including but not limited to ethanol, methanol and butanol) produced by the microbial fermentation of simple sugars and starches has already occurred. The fermentable substrate material is commonly referred to as “feedstock”. Commercial biofuel grade alcohol production can utilize feedstocks of simple sugars and starch sources including seeds (including but not limited to corn seed, wheat seed) as well as high sugar or simple starch content plant materials such as sugar beets, molasses, and sugar cane extracts. Feedstocks under evaluation for alcohol fermentation substrate also include the remaining parts of the plant and waste plant materials such as woody portions, husks, seed coatings, leafy materials, roots, fibrous material. These substrates are sometimes referred to as lignocellulosic, biomass, or cellulosic feedstocks. Still other fuel types include flammable gasses such as biogas, biohydrogen, etc.
There has been an increasing trend to rely on microbial fermentation activities in converting biomass into burnable fuels. The general process involves hydrolysis of the biomass (physical, chemical, and/or enzymatic) into fermentable substrates, and then microbes (including microalgae, fungi, and bacteria) ferment the substrates into biofuels. Another microbial source of biofuels is the production of lipids, especially triacyglycerides (TAG) by oleaginous microorganisms, including algae and fungi. Oil producing microorganisms are those that produce lipids and oils that can be converted into fuel quality lipids and oils. Of particular note is the production of biodiesels by algae.
Any biofuel generating process that utilizes microbial activity is subject to production slowdowns, failures, or reduction in production efficiencies through the activity of undesirable or contaminating microorganisms. The desired, biofuel producing organisms may be fungal, algal, including all clades of algae regardless of taxonomic position, or protozoan and the biofuel product may be alcohols, lipids and oils, or gas.
As a common form of biofuel, bioethanol is being widely used in many countries as motor fuels. In the U.S., fuel ethanol production has increased from 1.7 billion gallons in 2000 to almost 12.5 billion gallons in 2009 (www.ethanolrfa.org/pages/statistics). The number of ethanol fermentation facilities is also rapidly increasing, from 110 U.S. plants operating in 2007 to 187 in 2010. Bioethanal fermentation facilities utilize microbial activities to convert agricultural feedstocks into ethanol. The majority of commercial bioethanol fermentation plants in the U.S. are designed to utilize a grain feedstock, primarily corn, which is fermented by microorganisms, especially yeast, into ethanol. In standard operation, the complex carbohydrate chemistry of the feedstock is converted into simpler sugars by a combination of enzymatic (e.g. amylase or other starch-hydrolyzing enzyme) and/or physical (e.g. temperature and shearing) and/or chemical (e.g. by treatment with dilute sulfuric acid or other chemicals) treatment, forming a liquefied mash. Simple sugars in the liquefied mash are then used as substrates for ethanol fermentation by yeast. Cellulosic and lignocellulosic feedstocks are an attractive alternative to grain feedstocks, although they present additional challenges in terms of preparing the fermentable substrate. Because grain feedstock fermentation facilities utilizing yeast for ethanol production almost exclusively comprise current commercial operations, bacterial problems at these facilities are used in this application as illustrative examples of the method herein disclosed, and comprise a preferred aspect of this invention.
Example of the Target: Unwanted Bacteria in Ethanol Production
Chronic and acute bacterial contamination of the fuel ethanol fermentation process is common. Bacteria may initially enter the process with the feedstock or be present at the facility, for example on equipment, in liquids or in biofilms that serve as reservoirs for the bacteria. Bacteria may persist in the fermenters, along piping turns, and in heat exchangers and valves. While bacterial levels vary during the different steps for preparing the grain substrate for fermentation, by the time the processed mash is ready for yeast inoculation, the total bacterial levels in a normal, “healthy” fermentation facility are around 106 colony forming units (CFU) per ml in a wet mill and as high as 108 CFU/ml in a dry-grind facility (Skinner and Leathers 2004). However, bacterial levels higher than this frequently develop, negatively impacting ethanol yields. The most widely cited agents responsible for fuel ethanol fermentation slowdown are lactic acid bacteria (LAB), primarily members of the Gram-positive genera Lactobacillus, Pediococcus, Leuconostoc and Weissella (Bischoff, Liu et al. 2009). Bacteria inhibit the yeast fermentation process through the competitive consumption of sugars, which bacteria convert into organic acids instead of ethanol. These organic acids, primarily lactic and acetic, are inhibitory to the vitality of the yeast. Infections may be chronic, resulting in an overall constant loss of production efficiency, or acute, resulting in stagnated—or “stuck”—fermentation that requires the system be shut down for decontamination. Depending on the feedstock, fermentation system employed, and the nature of the contaminant, estimates on ethanol losses range from 1% for chronic infections to over 20% for extreme stuck fermentations (Bischoff, Liu et al. 2009, Makanjuola, Tymon et al. 1992, Narendranath, Hynes et al. 1997). Even a 1% decrease in ethanol yield is significant to ethanol producers (Narendranath 2003). At an average 50 million gallons per year (mgy) plant, a 1% loss equates to a decrease of 500,000 gallons of ethanol per year. Based on an average spot price of $1.84 per gallon (average for 2010, data available at www.neo.ne.gov/statshtml/66.html), this represents a loss of $920,000 in annual revenue.
While more data is available on the impact of bacteria on grain feedstock utilizing facilities, pilot plants utilizing lignocellulosic or biomass feedstock are also subject to contamination by undesirable bacteria (Schell, Dowe et al. 2007). It is anticipated that as more biomass and lignocellulosic alcohol fermentation facilities become operational, issues with bacterial contamination will also manifest. Additionally, biodiesel production using oleaginous algal or fungal or protozoan cultures are already known to be subject to production slowdowns due to invasive bacteria.
Regardless of the biofuel being generated, biofuel production can be negatively impacted by the activity of unwanted, invasive or contaminating bacteria. The scope of this invention covers all forms of biofuel production regardless of chemistry of the biofuel product or identity of the biofuel-producing organisms. Any biofuel production process that is negatively impacted by contaminating bacteria is covered by the scope of the invention. However, due to the abundance of information on ethanol production in corn fermentation facilities, this will be the example used to illustrate the method.
Control of Unwanted Bacteria: The LAB in Corn Ethanol Fermentation
Current Control Methods
Bacterial control methods have an immediate positive impact and even a simple one-log reduction in the amount of LAB can increase ethanol yield by approximately 3.7% (Bischoff, Liu et al. 2009). Bacterial contamination in fuel ethanol plants is typically controlled by a combination of plant management approach and through the addition of chemical antimicrobials and antibiotics. The types and amounts of chemicals that can be used to control LAB are limited because the compounds must reduce bacteria without affecting the yeast culture and must also not carry over as harmful residue in the solid co-products of fuel ethanol fermentation, which is frequently sold as distillers dried grains with solubles (DDGS) for animal feeds. The plant management approach involves the routine cleaning of equipment and reactors, as well as controlling physical and chemical parameters such as temperature, pH, and acid levels to favor yeast over bacterial growth. Chemical antimicrobials that can be added to reduce bacterial levels include typical quaternary compounds and gluteraldehyde, as well as more specialized formulations such as a stabilized ClO2 product sold by DuPont under the trade name FermaSure™.
Challenges Associated with Antibiotic Use
Not surprisingly, antibiotics, in particular virginiamycin and penicillin, have been found particularly effective in curbing bacterial populations without disturbing the yeast. This has led to the widespread use of antibiotics in the fuel ethanol fermentation industry. However, antibiotic residue has been detected in the solid distillers grain residue (DG) that is sold as livestock feed (De Alwis and Heller). Additionally, there is evidence that antibiotic use leads to selection for antibiotic resistance (Bischoff, Skinner-Nemec et al. 2007). Even though effective, it is generally agreed that there needs to be an end to indiscriminate, non-therapeutic use of antibiotics. Thus, the ethanol industry in particular, and the biofuel industry in general, needs to move quickly to replace antibiotics.
Bacteriophage Control of Unwanted Bacteria
Bacteriophage, or phage, are the viral predators and parasites of bacteria. Included in this definition are the dsDNA (double stranded DNA genome) tailed phages, referred to as the Caudovirales, or sometimes the caudoviruses, caudophage or tailed phage. Among the tailed phages included here are members of the three morphotypes, including the contractile tailed Myoviridae, the long non-contractile tailed Siphoviridae, and the short tailed Podoviridae. It should be noted that these morphological distinctions do not reflect phylogenetic relationships based on genetic analysis and so no implication of relatedness in inferred by this classification system. Also included in the definition of phage are the phages classified based on genome composition, morphology, and presence or absence of a lipid envelope, including the Tectiviridae, Corticoviridae, Lipothrixviridae, Plasmaviridae, Rudiviridae, Fuselloviridae, Inoviridae, Microviridae, Leviviridae, and Cystoviridae. As taxonomic classifications are frequently updated based on new techniques, such as molecular data, these definitions of phages includes any new phage families that might be created. It should also be noted that these phage families includes members often referred to as viruses of Archaea.
Historical and Current Commercial Phage Use
The bacteriolytic nature of phages leads to interest in their use as antimicrobials. Phage themselves are not new, having been discovered during the First World War. The most obvious use of phages is for medical applications. While early interest in phage therapy was suppressed by the introduction of antibiotics, the recent rise in antibiotic resistance and costly food contamination events has led to a resurgence of interest in phages (Kropinski 2006; Mattey and Spencer 2008; Housby and Mann 2009). In the United States, phages have been applied on human patients as part of a more comprehensive approach to controlling and curing chronic wounds associated with diabetic ulcers and pressure wounds. The commercial development of phages to treat infections in humans is crippled by the expensive regulatory requirements for new drug approval. This problem is exacerbated by the ambiguous classification of phages within the context of drug testing protocols. In contrast, the application of phages in food and agriculture faces fewer challenges and many applications are under investigation (Sabour and Griffiths 2010). In 2007, phages were approved by the FDA as a food additive, specifically for the control of the food-borne pathogen Listeria on commercial luncheon meats (Bren 2007). Commercial phage products sold in the U.S. include AgriPhage, sold by Omnilytics and designed to control Xanthomonas infestations in peppers and tomatoes and Finalyse, sold by Elanco Foods and designed to control E. coli 0157:H7 levels on slaughterhouse cattle.
Advantages of Phages for Use in Biofuel Production
Phages are natural, ubiquitous bacteriolytic agents with extremely high host specificity. Phage formulations and antibiotics both have advantages over chemical biocides in that they specifically kill target unwanted host bacteria without interacting with non-bacterial microorganisms (such as yeast or algae) responsible for alcohol or oil production. In contrast, chemical biocides are much less selective and doses effective against bacteria may adversely modulate growth of the biofuel producing organisms. Thus, the present innovative application of phages to control unwanted bacteria in the biofuel production process will lead to both immediate economic and long-term socioeconomic impacts.